Antibodies, also known as immunoglobulins (Ig), are essential components of the adaptive immune response of vertebrates. They are found in blood plasma or other bodily fluids, for example on mucosal areas performing recognition and neutralization of antigens. Antigens are mostly foreign substances such as viruses, bacteria or other pathogenic substances (toxins) invading the vertebrate body (“non-self” antigens), but sometimes these can also be autoantigens which are derived from molecules occurring in the body (“self” antigens).
The IgG antibody, the predominate antibody type in humans, is composed of four polypeptide chains: two identical heavy chains (HC) of about 50 kDa and two identical light chains (LC) with an approx. molecular weight of 25 kDa. While the isotype is specified by the kind of heavy chain, there also exist different light chains: λ (lambda) and κ (kappa).
Within the variable domains three regions show an especially high degree of variability: they are known as hypervariable or complementarity determining regions (VL CDR1-3 and VH CDR1-3) and alternate with four quite invariant framework regions (FR1-4). As a result of Ig domain folding the six CDR loops from VL and VH are in close spatial proximity and can interact with a certain region on the antigen (epitope) by different non-covalent interactions such as hydrogen bonds, van der Waals forces and ionic interactions.
Besides full-length antibodies, fragments of them have been developed. The most common antibody fragments are Fab (fragment antigen binding) and scFv (single chain fragment variable). The Fab fragment is composed of antibody light chain (VL and CL) connected by a disulfide bond to a part of the heavy chain (VH and CH1). In contrast, the scFv fragment consists of variable domains of heavy and light chain fused by a short peptide linker. Fab fragments can be produced by proteolytic cleavage of an immunoglobulin with the enzyme papain, which cleaves the molecule in the hinge region and produces consequently two Fab fragments and one Fc fragment (fragment crystallizable). Today Fab fragments, as well as many other antibody fragments, are mainly generated via recombinant DNA technologies. Besides Fab and scFv, there exist so far a wide range of different antibody fragments formats such as dsFv, single domain fragments or diabodies with two possible specificities.
For the development of novel antibody specificities genetic information of whole antibody repertoires can be amplified and used for the generation of gene libraries, which are subsequently selected to identify single antibodies with desired properties. The success of this approach basically depends on size (complexity) and quality of the generated libraries. In general libraries can be divided into three different types: immune libraries from immunized animals or infected humans, naïve libraries from non-immunized donors and synthetic/semi-synthetic libraries constructed with degenerated oligonucleotides. Besides source of antibody repertoire, these libraries differ in required library size. In contrast to naïve or synthetic approaches, immune libraries require rather low complexities, because previous immunizations supply antibody genes after in vivo affinity maturation and increase the chance to isolate an antibody with desired specificity. Nevertheless, naïve or synthetic libraries are favorable in the generation of antibodies against for example self-antigens or non-immunogenic haptens, because they overcome immunological tolerance mechanisms.
The generation of recombinant antibodies based on antibody gene libraries requires strong and efficient selection systems, for example phage display. This selection system deals with the presentation of molecules on the surface of filamentous bacteriophages such as M13. Filamentous phages are viruses infecting Gram-negative bacteria such as, for example, E. coli via F-pili; the infection does not lead to cell lysis but to continuous release of assembled phage particles from infected cells.
The human CD8 molecule is a glycoprotein and cell surface marker expressed on cytotoxic T-cells (CTLs). These are a subset of T-lymphocytes and play an important role in the adaptive immune system of vertebrates. They are responsible for the elimination of virus-infected cells or other abnormal cells such as some tumor cells. These cells are specifically recognized via the T-cell receptor (TCR), which interacts with the certain antigen presented via MHC (major histocompatibility complex) class I on target cells.
In many protein interactions metal ions play an important role. About 20% of all antibody crystal structures contain metal ions, although most of these may be crystallization artifacts (Zhou et al., Proc Natl Acad Sci USA 102, 14575-14580 (2005)). There are publications describing antibodies with metal specificities, for example against chelated copper ions (Kong et al., Biol Trace Elem Res 145, 388-395 (2012)) or catalytic active metallo-antibodies (Roberts and Getzoff, FASEB J 9, 94-100 (1995)). Metal ions can also influence a certain conformational state in an antigen, which is selectively recognized by an antibody, for example a Ca2+ sensitive epitope in human factor IX (Bajaj et al., Proc Natl Acad Sci USA 89, 152-156 (1992)). Furthermore, the monoclonal M1 antibody directed against the FLAG peptide has been claimed to bind its antigen in a calcium dependent manner, allowing a release in immunoaffinity purifications with chelating agents such as EDTA (Hopp et al., Mol Immunol 33, 601-608 (1996)). However, M1 does not show any affinity differences of antibody-antigen interaction in the presence or absence of Ca2+, so this observation results in the assumption that the added EDTA forms a short transition state with less affinity and there is no real calcium dependency (Einhauer and Jungbauer, J Chromatogr A 921, 25-30 (2001)). In contrast to these findings, two publications determined the crystal structure of two calcium dependent antibodies which show calcium ions at the interface of antigen and antibody interaction (Zhou et al., Proc Natl Acad Sci USA 102, 14575-14580 (2005); and Wojciak et al., Proc Natl Acad Sci USA 106, 17717-17722 (2009)). First, Zhou et al. disclosed the calcium dependent human CD4 reactive monoclonal antibody Q425. The binding of this antibody was affected by Ca2+ ions and to a lesser extent by Sr2+ and Cd2+ ions, but not or nearly negligible by Mg2+, Ba2+, Mn2+, Cu2+, Co2+, Ni2+, Zn2+ and K+ ions. In surface plasmon resonance experiments, a 55,000-fold affinity enhancement in the presence of 25 mM Ca2+ compared to calcium-free conditions was determined. The protein structure revealed that a single calcium ion is primarily coordinated by negatively charged side chains of the amino acids aspartate and glutamate in the CDRs of the light chain. Besides these observations, Wojciak et al. disclosed the crystal structure of the α-sphingosine-1-phosphate (S1P) LT1009 protein in complex with its antigen, a lipid (non-protein), wherein LT1009 is a papain-generated Fab fragment of a humanized antibody. The structure showed an association of calcium ions with mainly glutamate side chains of CDRs of the light chain. A 100-fold change in affinity when calcium was present or absent was also reported. In both cases (Q425 and LT1009) calcium ions are bridging the interaction between antigen and antibody at their interface. Due to the Ca2+ dependency the binding reaction of these antibodies to their binding partners such as a protein or a lipid is reversible under mild conditions, for example by the addition of EDTA. This property was generated accidentally and not intentionally; in both cases standard methods for generating antibodies were used without the intention to generate calcium ion dependent antibodies or antibody fragments.
In Erasmus (Thesis, San Diego State University (2012)) LT1002 is disclosed which is the murine pre-engineered antibody version of LT1009. LT1002 was analyzed and showed a tendency to bind Ca2+ ions and to a smaller extent also Mg2+ and Ba2+ ions. This is not surprising since LT1002 is the pre-version of LT1009. The amino acid sequence of LT1002 is disclosed in U.S. Pat. No. 8,067,549.
Fanning et al. (Biochemistry 50, 5093-5095 (2011)) developed a synthetic library approach to generate a novel dual-specific antibody. Using a combinatorial histidine-scanning phage display library, potential metal binding sites were introduced throughout an anti-RNase A antibody interface. Stepwise selection of RNase A and metal binding produced a dual-specific antibody that retained near wild-type affinity for its target antigen while acquiring a competitive metal binding site for nickel that is capable of controlling the antibody-antigen interaction. The publication does not disclose if this approach using introduced histidine residues in the antibody/antigen interface also works with metal ions other than Ni2+, because the authors showed that the metal site is specific for nickel as titrations performed in the presence of calcium ions did not show any appreciable change in the observed binding constant. Another major limitation of this approach is the fact that the resulting antibody shows the highest affinity to its antigen in the absence of metal ions, while addition on Ni2+ partially decreases the affinity, excluding this approach for the generation of antibodies which bind their antigen in the presence of a metal ion and do not bind or release their antigen in the absence of the metal ion.
Using a similar approach, Murtaugh et al. (Protein Sci 20, 1619-1631 (2011)) used a combinatorial histidine library to introduce histidine residues into the binding interface of an anti-RNase A single domain VHH antibody. They generated a “switchable” antibody variant that binds to its antigen in a pH-dependent manner. Although such antibody variants can be useful for the controlled binding to its antigen in one situation (such as pH>7) and the controlled dissociation from the antigen in another situation (such as pH<5), the publication does not disclose how “switachble” antibodies can be generated which can be controlled with metal ions. There is also the disadvantage that these pH-dependent antibodies are not compatible with the strict requirements of cell culture conditions where cells are usually kept within a narrow pH range at nearly neutral pH.
U.S. Pat. No. 6,111,079 discloses a metal binding protein which selectively binds a complex of a heavy metal, such as lead cation, and glutathione. Mice were immunized with a glutathione/Pb2+ complex, and monoclonal antibodies were generated by hybridoma technology. These antibodies were screened against their ability to bind a glutathione/Pb2+ complex. The disclosure is directed to methods for detecting, removing, adding, or neutralizing heavy metals in biological and inanimate. These proteins have a high discrimination for lead cations and against other metallic cations. U.S. Pat. No. 6,111,079 does not teach which amino acids are de facto involved in lead cation binding. The method described in U.S. Pat. No. 6,111,079 has the disadvantage of generating only antibodies against a glutathione/Pb2+ complex. Such a heavy metal binding antibody also has the disadvantage that it is not compatible with the strict requirements of cell culture conditions due to toxicity of the heavy metal Pb2+.
The frequency of metal ion binding proteins which bind their binding partner such as an antigen in the presence of the metal ion and which can be released from the binding partner by removal or complexation of the metal ion by for example EDTA or EGTA seems to be rather low. For antibodies, Zhou et al. (2005) speculated that although ˜20% of antibody crystal structures contain metal ions most of these are crystallization artifacts, and in other cases partially coordinated metal ions offer no or little advantage in terms of binding. Instead, for an antibody, a direct antigen binding usually seems to be preferred over interfacial metal coordination.
There is a need in the art for the targeted generation of metal ion binding proteins such as antibodies or fragments thereof in which metal ions such as Ca2+ are bridging the interaction between the protein, e.g. an antibody, and the binding partner of the protein, e.g. an antigen, at their interface. This results in a binding between the metal ion binding protein and the binding partner of the protein which is reversible, depending on the presence or absence of the metal ion or a chelating or complexing reagent such as EDTA or oxalate, respectively.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.